Analyte sensor

ABSTRACT

Matrix materials, such as sol-gels and polymers derivatives to contain a redox active material can be used to form electrodes and probes suitable for use in pH meters and other analyte sensing devices.

RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.13/812,135, filed Mar. 19, 2013, entitled ANALYTE SENSOR which is a U.S.National Stage of International Application No. PCT/US2011/045385, filedJul. 26, 2011, and entitled ANALYTE SENSOR which claims the benefit ofU.S. Provisional Application No. 61/367,590, filed Jul. 26, 2010. Thisapplication claims priority to and incorporates herein by reference theabove-referenced applications in their entirety.

BACKGROUND OF THE INVENTION

The present invention relates generally to technology for detecting ananalyte. In various embodiments, the invention relates to devices formeasuring pH, the potential of hydrogen, which is a measure of theacidity or alkalinity of a solution. The pH of a solution is determinedby the concentration of dissolved hydrogen ions (H⁺) (also referred toas hydronium ions, H₃O⁺) within the solution. As the concentration ofdissolved hydrogen ions within the solution increases, the solutionbecomes more acidic. Conversely, the solution becomes more basic as theconcentration of dissolved hydrogen ions within the solution decreases.The concentration of dissolved hydrogen ions within a solution hastraditionally been measured with a glass electrode connected to anelectronic meter that displays the pH reading. Traditionally the terms“probe” and “electrode” have been used interchangeably to describe afunctional grouping of component electrodes. As used herein, the term“electrode” is used to refer to a specific electrode in a probe, i.e.,such as a “working electrode”, a “reference electrode”, or a “counterelectrode”, and “probe” refers to a functional grouping of electrodessufficient to generate a signal that can be processed to generate areading indicative of the concentration of an analyte of interest in asolution.

The traditional glass pH probe has a working electrode (WE) that is anion-selective electrode made of a fragile, doped glass membranesensitive to hydrogen ions. The pH-responsive glass membrane is theprimary analyte sensing element in this type of probe and so is referredto as the “working” electrode. Hydrogen ions within the sample solutionbind to the outside of the glass membrane, thereby causing a change inpotential on the interior surface of the membrane. This change inpotential is measured against the constant potential of a conventionalreference electrode (RE), such as an electrode based on silver/silverchloride. The difference in potential is then correlated to a pH valueby plotting the difference on a calibration curve. The calibration curveis created through a tedious, multistep process whereby the user plotschanges in potential for various known buffer standards. Traditional pHmeters are based on this principle.

The response of traditional glass working electrodes (and probes andmeters containing them) to pH is unstable, and glass probes periodicallyrequire careful calibration involving tedious, time-consuming processes,multiple reagents, and a well trained operator. The special propertiesand construction of the glass probes further require that the glassmembrane be kept wet at all times. Thus, routine care of the glass proberequires regular performance of cumbersome and costly storage, rinsing,cleaning, and calibration protocols performed by a well trained operatorto ensure proper maintenance and working performance.

In addition to tedious maintenance and storage requirements, traditionalglass probes are fragile, thereby limiting the fields of application ofthe glass probe. In particular, the fragile nature of the glass probemakes it unsuitable for use in food and beverage applications, as wellas use in unattended, harsh, or hazardous environments. Accordingly,there is a need in the art for pH probes and meters (as well as otheranalyte probes and meters) that address and overcome the limitations oftraditional pH probes and meters employing the glass probe.

In response to the limitations described above for traditional glassprobe pH measuring systems, voltammetric systems were proposed to offera more robust system for the determination of pH. In a voltammetricsystem, an electrical potential is applied in a controlled manner,typically varied linearly with time, and the corresponding currentflowing through a conductive material is monitored by means of, forexample, a potentiostat (see, for example, Wang, “AnalyticalElectrochemistry,” 3^(rd) ed, John Wiley & Sons, 2006). Initialproposals (see U.S. Pat. No. 5,223,117) were based on the concept of aWE composed of a conductive substrate with a redox active moleculeattached to its surface. The hypothesis was that, provided anappropriate “analyte-sensitive”, redox active material (ASM) was used,the potential at which the maximum current flows in this system would bea function of the pH of the analyte solution. However, this initialproposal met with little enthusiasm, perhaps because it was demonstratedwith an electrode that used gold as a substrate.

Significant advances were made in both theory and research laboratorypractice of voltammetry-based analyte sensing systems when researchersdiscovered that carbon could replace gold as the conductive substrateand, moreover, that, regardless of the substrate, mixtures of redoxactive materials could be used in voltammetric systems (see PCT Pub.Nos. 2005/066618 and 2005/085825). One particularly intriguing proposalby these researchers was that a mixture of “analyte-sensitive” materials(ASMs) and “analyte-insensitive” materials (AIMs) could be attached to aconductive substrate and effectively convert it into both a WE (signalgenerated by the ASM) and a reference electrode (RE) (signal generatedby the AIM). No significant advances, however, in either theory orpractice were made for some time after these initial proposals andresearch (see, e.g., PCT Pub. Nos. 2007/034131 and 2008/154409).

The next significant advance in the field occurred when scientistsdiscovered that, in practice, no redox active material is completely“analyte-insensitive” and that practical application of voltammetrictechnology should focus on WEs without AIMs. These scientists alsodiscovered, however, that, regardless of whether a redox active materialwas characterized as an ASM or AIM, it could be made trulyanalyte-insensitive by sequestration in an ionic medium. This discoveryled to the analyte-insensitive electrode or AIE, which could not only beused as a replacement of the conventional RE in traditional pH measuringsystems but could also be used with WEs based on voltammetry. See PCTPub. No. 2010/104962. Soon after these discoveries, pH meters suitablefor use on the laboratory benchtop and for important research anddevelopment applications were created. See PCT Pub. Nos. 2010/111531 and2010/118156.

However, despite these highly promising advances, in practice, theuseful lifetime of, and suitable applications for, these probes weredependent, among other factors, on the means by which the redox activemolecules were affixed to the substrate surface of the electrode. Forexample, the two main approaches for attaching the redox active moleculeto the substrate of the electrode, non-covalent adsorption and directchemical (covalent) linkage, both exhibit rapidly decaying signals, somelosing much of their functionality in less than two days, andinstability in certain analyte solutions or environmental conditions.

There is, therefore, a need in the art for methods and compositions forfixing redox active materials to the conductive substrate of anelectrode for use in a voltammetry-based analyte-sensing system and forelectrodes, probes, pH meters, and other analyte sensing devices basedon voltammetric systems that provide longer useful lifetimes and can beused for a wider variety of applications. The present invention meetsthese needs.

SUMMARY OF THE INVENTION

The present invention relates generally to methods for immobilizing aredox active material to a conductive substrate, compounds, andcompositions useful in the method, electrodes produced by the method,and pH meters and other analyte sensing devices incorporating one ormore electrodes of the invention.

In one aspect, the invention relates to methods for immobilizing redoxactive compounds in matrix materials that can be coated on the surfaceof a suitable substrate or molded to form a redox active surface for usein analyte-sensing electrodes, probes, and sensors, such as pH metersand other analyte-sensing devices. The method is generally applicable toany redox active compound, but in many embodiments, is practiced usingthe AIMs or ASMs known to be useful in voltammetry-based analyte-sensingmethodology. In the method, the AIM or ASM is covalently attached to thepolymer that forms the matrix. In some embodiments, the AIM or ASM isfirst attached to a monomer unit that is then polymerized to form thematrix material. In other embodiments, the AIM or ASM is attached to apolymer that may be used directly or cross-linked and then used.Variations and combinations of these embodiments are also provided.

In another aspect, the invention relates to electrode components,electrodes, probes, and meters comprising one or more matrix materialsof the invention. In one embodiment, the invention provides a workingelectrode that contains a matrix material of the invention comprising anASM coated on an electrically conductive substrate such that it remainsin electrical contact with the substrate. The present invention alsoprovides sensors such as pH meters and other analyte sensing devicescomprising such WEs. In some embodiments, the matrix material in theseWEs can also have one or more AIMs attached to them. In anotherembodiment, the invention provides an AIE that contains a matrixmaterial of the invention comprising either an ASM or AIM or both. Inany of these embodiments, the matrix material of the invention can becoated onto the surface of a distinct electrically conductive substrateto form an electrode (or component thereof) or can be molded to form theelectrode (or component).

In another aspect, the present invention relates to compounds,compositions, and methods for the formation of the matrix materials ofthe invention. In one embodiment, the invention provides compounds,compositions and methods for forming sol-gel matrix materials. Inanother embodiment, the invention provides compounds, compositions andmethods for forming other polymeric matrix materials.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart demonstrating a comparison of signal sensitivitybetween physically adsorbed ASM WEs and sol-gel derived WEs inaccordance with a representative embodiment of the present invention.

FIG. 2 is a chart demonstrating the peak position of an ASM containingsol-gel derived WE in accordance with a representative embodiment of thepresent invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds, compositions, methods,electrodes, and sensors, including solid state analyte sensors superiorto those currently known in the art. Specifically, in one embodiment,the present invention provides a solid state analyte sensor systemincorporating a redox active (analyte sensitive or analyte insensitive)material within a silane-based sol-gel matrix material or an organicpolymeric matrix material. Accordingly, some embodiments of the presentinvention provide improved analyte-dependent signals exhibiting morewell-defined peaks, greater peak position stability, higher peakintensity, increased peak longevity, and longer useful lifetime for theelectrode construct, relative to currently available analyte sensorsystems. Some embodiments of the sensor components, as well asconfigurations and compositions of those components, are described indetail below, following definitions provided for the convenience of thereader.

Definitions

As used in the specification and the appended claims, the singular forms“a,” an” and “the” include plural referents unless the context dictatesotherwise. Thus, for example, reference to “a binder” includes mixturesof binders, and a reference to a conductive material may include morethan one such binder.

“Alkanyl” refers to a saturated branched, straight-chain or cyclic alkylgroup. Typical alkanyl groups include, but are not limited to, methanyl;ethanyl; propanyls such as propan-1-yl, propan-2-yl (isopropyl),cyclopropan-1-yl, etc.; butyanyls such as butan-1-yl, butan-2-yl(sec-butyl), 2-methyl-propan-1-yl (isobutyl), 2-methyl-propan-2-yl(t-butyl), cyclobutan-1-yl, and the like.

“Alkenyl” refers to an unsaturated branched, straight-chain or cyclicalkyl group having at least one carbon-carbon double bond derived by theremoval of one hydrogen atom from a single carbon atom of a parentalkene. The group may be in either the cis or trans conformation aboutthe double bond(s). Typical alkenyl groups include, but are not limitedto, ethenyl; propenyls such as prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl (allyl), prop-2-en-2-yl, cycloprop-1-en-1-yl;cycloprop-2-en-1-yl; butenyls such as but-1-en-1-yl, but-1-en-2-yl,2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl,buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl,cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, and the like.

“Alkoxy” by itself or as part of another substituent refers to a radical—OR₁₀₀ where R₁₀₀ represents an alkyl group as defined herein.Representative examples include, but are not limited to, methoxy,ethoxy, propoxy, butoxy, and the like.

“Alkyl” refers to a saturated or unsaturated, branched, straight-chainor cyclic monovalent hydrocarbon group derived by the removal of onehydrogen atom from a single carbon atom of a parent alkane, alkene oralkyne. Typical alkyl groups include, but are not limited to, methyl;ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl,propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl,prop-2-en-1-yl (allyl), cycloprop-1-en-1-yl; cycloprop-2-en-1-yl,prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butyls such as butan-1-yl,butan-2-yl, 2-methyl-propan-1-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl,but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl,but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl,but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, and the like. The term“alkyl” is specifically intended to include groups having any degree orlevel of saturation, i.e., groups having exclusively singlecarbon-carbon bonds, groups having one or more double carbon-carbonbonds, groups having one or more triple carbon-carbon bonds and groupshaving mixtures of single, double and triple carbon-carbon bonds. Wherea specific level of saturation is intended, the expressions “alkanyl,”“alkenyl,” and “alkynyl” are used. The expression “lower alkyl” refersto alkyl groups comprising from 1 to 8 carbon atoms.

“Alkynyl” refers to an unsaturated branched, straight-chain or cyclicalkyl group having at least one carbon-carbon triple bond derived by theremoval of one hydrogen atom from a single carbon atom of a parentalkyne. Typical alkynyl groups include, but are not limited to, ethynyl;propynyls such as prop-1-yn-1-yl, prop-2-yn-1-yl, butynyls such asbut-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, and the like.

An “analyte” is a chemical species of interest present in a sample, thepresence of which is detectable or the concentration of which ismeasurable by using an analyte sensor system that incorporates a workingelectrode.

An “analyte insensitive electrode” (AIE) is a special case of areference electrode where the current flow depends in part on redoxprocesses that are independent of the presence or concentration ofspecies (apart from a minimum threshold of supporting electrolyte) inthe sample composition including but not limited to the analyte. The AIEserves to provide a response that does not vary across time or samplecomposition and therefore can be used as an internal standard or ‘zeropoint’ to which the WE response may be compared. See PCT Pub. No.2010/104962, incorporated herein by reference. An AIE contains one ormore ASMs or AIMs in electrical contact with a conductive substrate, apseudo reference electrode (PRE) as defined below, and a means to placethe ASM or AIM and the PRE in a constant chemical environment isolatedfrom, but in electrical and fluid communication with, an analytesolution. As used herein, AIE refers to the integrated functional unit(ASM or AIM, PRE, and other components of the AIE), although it will beclear from the context that, in some of these references, the matrixmaterial component is the subject of the disclosure.

An “analyte-insensitive material” or “AIM” is a redox-active materialthat is insensitive or substantially insensitive to the presence or theconcentration of an analyte in a sample. “Substantially insensitive” toan analyte is used to mean insensitive within the tolerances requiredfor a given application, as those tolerances are defined by an end user.

An “analyte sensing device” is a sensor, a means to measure the signalfrom the sensor, and optionally a means to display that signal. A pHmeter is a type of analyte sensing device. Thus, in some embodiments, ananalyte sensing device includes a controller/processor unit, associatedprograms and algorithms, and a probe.

An “analyte-sensitive material” or “ASM” is a redox-active material thatis sensitive or substantially sensitive to the presence or concentrationof an analyte in a sample within those user-defined application-specifictolerances. “Substantially sensitive” to an analyte is used to meansensitive within the tolerances required for a given application, asthose tolerances are defined by an end user. An ASM can function as anAIM by sequestration in a suitable ionic medium, as in an AIE.

“Aryl” refers to a monovalent aromatic hydrocarbon group derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. Typical aryl groups include, but are not limitedto, groups derived from aceanthrylene, acenaphthylene,acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene,fluoranthene, fluorene, hexacene, hexaphene, hexalene, as-indacene,s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene,ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene,rubicene, triphenylene, trinaphthalene, and the like. The aryl group maybe, for example, (C₅-C₁₄) aryl, including but not limited to (C₅-C₁₀).Illustrative aryls include cyclopentadienyl, phenyl and naphthyl.

“Arylalkyl” refers to an acyclic alkyl group in which one of thehydrogen atoms bonded to a carbon atom, typically a terminal or sp³carbon atom, is replaced with an aryl group. Typical arylalkyl groupsinclude, but are not limited to, benzyl, 2-phenylethan-1-yl,2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl,2-naphthylethen-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl and thelike. Where specific alkyl moieties are intended, the nomenclaturearylalkanyl, arylakenyl and/or arylalkynyl is used. In preferredembodiments, the arylalkyl group is (C₆-C₂₀) arylalkyl, e.g., thealkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C₁-C₆) andthe aryl moiety is (C₅-C₁₄). Illustrative embodiments include thearylalkyl group (C₆-C₁₃), e.g., the alkanyl, alkenyl or alkynyl moietyof the arylalkyl group is (C₁-C₃) and the aryl moiety is (C₅-C₁₀).

“Coaxial” refers to a common axis about which various components, forexample, electrodes, are positioned. In some embodiments, “coaxial”refers to a radial symmetry of concentrically or approximatelyconcentrically positioned components. In some embodiments, the term“coaxial” refers to one or more electrodes being concentricallypositioned within an outer or externally positioned electrode component;for example and without limitation, a WE, CE, and RE are coaxiallypositioned when the CE is the outer ring of a sensor tip that isimmersed in the analyte solution, the WE is in the middle of the tip,and the RE is interposed between CE and the WE. See PCT Pub. No.2010/111531, incorporated herein by reference.

A “counter-electrode” or “CE,” also sometimes referred to as an“auxiliary electrode” is an electrode that is required, in some analytesensors, to pass current through the cell to complete the electricalcircuit. This electrode simply serves as a source or sink of electronsand allows current to flow through the WE to effect the redox reaction.To avoid unwanted electrochemical redox processes occurring at the CE,which may interfere with the signal measured at the WE, CEs aretypically made using relatively chemically inert materials, commonlyplatinum (Pt), but carbon allotropes are also commonly employed.

“Dispersed” or “associated” in reference to a material, means that it isdissolved in a solution or colloidally suspended in a gas, liquid orsolid. The term also encompasses embodiments in which the material iscovalently bound to the surface of a solid or to a component of thesolid. The term also encompasses embodiments in which the material isincorporated as a dopant in a crystal lattice. The term also encompassesmaterials intercalated within a solid.

An “electrode” is a component of a probe.

A “pseudo-reference electrode” (PRE) refers to a type of referenceelectrode that has traditionally been used in non-aqueous electrolytes.These electrodes typically do not comprise both halves of a redox coupleand are therefore not well-defined reference electrodes of fixedcomposition and potential. However, they provide a reasonably constantpotential over the timescale of an electrochemical experiment (on theorder of minutes), and the absolute potential of the PRE can then becalibrated back to a RE if required. One example of a PRE is a silverwire (used commonly in non-aqueous electrochemistry). More recently,PREs have been used as a component of an AIE.

A “redox-active material” is a compound or composition that may beoxidized and reduced. “Redox activity” refers to either or both of thoseprocesses.

A “reference electrode” (RE) is an electrode used to establish thepotential difference applied to the WE. Conventional REs have a certainfixed chemical composition and therefore a fixed electrochemicalpotential, thus allowing measurement of the potential difference appliedto the WE in a known, controlled manner. An RE typically comprises twohalves of a redox couple in contact with an electrolyte of fixedchemical composition and ionic strength. Because both halves of theredox couple are present and the composition of all the species involvedis fixed, the system is maintained at equilibrium, and the potentialdrop (i.e., the measured voltage) across the electrode-electrolyteinterface of the RE is then thermodynamically fixed and constant. Forexample a commonly used RE system is the Ag|AgCl|KCl system with adefined and constant concentration of KCl. The two half-cell reactionsare therefore: Ag⁺+e⁻→Ag; and AgCl+e⁻→Ag+Cl⁻. The overall cell reactionis therefore: AgCl→Ag⁺+Cl⁻ for which the Nernst equilibrium potential isgiven as: E=E₀−(RT/F)*ln[Cl⁻], where E is the measured RE potential, E₀is the standard potential of the Ag|AgCl couple vs. the standardhydrogen electrode with all species at unit activity (by convention thestandard hydrogen electrode is defined as having a potential of 0.0V);and R, T, and F are the universal gas constant, temperature, and Faradayconstant, respectively, in appropriate units. Hence, the potential ofthis system depends only on the concentration (more strictly speakingthe activity) of Cl⁻ ion present, which, if this is fixed, provides astable, fixed potential. Many other RE systems are known in the art. Itis imperative that the composition of the RE remains constant, and hencealmost no current should be passed through the RE (otherwiseelectrolysis will occur and the composition of the RE will change),which necessitates the use of a third electrode, the counter electrode(CE), to complete the circuit. However, two-electrode configurations canbe used in the special case where the WE is a microelectrode, having atleast one dimension typically smaller than 100 micrometers. In thiscase, the currents passed at the WE are small, and therefore atwo-electrode cell can be used with a RE, but without the need for a CE.

A “probe” refers to a sensor that contains multiple electrodes. A probecan include, for example, a working electrode, a counter-electrode and areference electrode (either a conventional reference electrode or apseudo reference electrode). A probe can include, for example, a workingelectrode, a counter electrode and an analyte-insensitive electrode.

A “sensor” is an electrode or collection of electrodes that generate asignal in response to the presence of an analyte.

A “surface” of an electrode refers to the functional surface, i.e., thatportion of the surface that is in contact with the analyte sample andserves an electrical or electrochemical purpose. It would not, forexample, include an insulating WE housing through which no current orvoltage passes. The surface of a WE is the portion of the electrodesurface in contact with the sample that detects current or electricalpotential relative to the RE. The surface of a CE refers to the portionin contact with the sample which serves to deliver or accept current toor from the WE.

A “working electrode” or “WE” is the electrode at which theelectrochemical process for detecting the analyte of interest occurs. Ina sensor, the working electrode may be sensitive to one or moreanalyte(s) in the test sample, or it may be chemically modified withanalyte sensitive species/materials. The electrochemical response of theworking electrode is measured after some perturbation to the systemunder study has been applied. For example, the perturbation may be theapplication of a potential difference to the WE which induces electrontransfer to occur, and the resulting current at the WE is then recordedas a function of the applied potential (voltammetric mode). This exampleof mode of operation is illustrative and not exhaustive, as many othermodes are known in the art. The WEs of the invention contain an ASM thatcan undergo a reversible electrochemical redox reaction dependent uponthe concentration of analyte (hydrogen ions for a pH meter; otheranalytes for other analyte sensing devices) in a sample solution and anapplied electrical potential. For example, where there is a highconcentration of hydrogen ions present in a sample solution, the redoxreaction occurs at a lower potential. Conversely, where there is a lowconcentration of hydrogen ions present in a sample solution, the redoxreaction occurs at a higher potential. The relationship between thesecharacteristic potentials and the sample solution pH is a function ofthe chemical identity of the ASM. An algorithm converts electricalpotential to pH value to provide a means of determining the pH of anunknown sample.

With the above definitions in mind, the reader can better appreciate thevarious aspects and embodiments of the invention described below.

In one important aspect, the present invention provides workingelectrodes for use in analyte sensing devices and matrix materials forthose WEs. In some embodiments, the matrix material of the currentinvention comprises an ASM covalently attached to a mechanically andchemically stable matrix material. In some embodiments, the matrixmaterial is attached to the substrate surface. In other embodiments, thematrix material alone serves as the electrode (i.e.: there is nosubstrate). In many embodiments, the matrix material is in the form of asurface coating (composed of a sol-gel or other polymeric material) on aconductive substrate, which together form a WE or AIE. In the case of aWE, the matrix material in this embodiment functions to attach the ASMcovalently to a solid structure (the conductive substrate), offerunhindered access of the analyte sample to the ASM, and provideefficient electrical connection between the ASM and the conductivesubstrate. In some embodiments, the matrix material contains more thanone ASM which differ in the potential at which the respective ASMs sensethe analyte. The advantage of this multiple ASM system is the greaterdegree of precision and accuracy associated with multiple data pointsbeing taken in the same measurement. Another advantage of this multipleASM system is its ability to maintain accurate sensing of the analyteconcentration even if certain components of the test sample interferewith the normal response of one of the ASMs.

Further, in some embodiments, the present invention provides a matrixmaterial (a sol-gel or polymeric material) that contains both an ASM andan analyte insensitive molecule (AIM). The advantage of this system isthat a separate reference electrode is unnecessary in the system,because the signal derived from the AIM is used for the point ofreference. As with the WE described immediately above, in someembodiments of this electrode, the matrix material contains multipleASMs as well as one or more AIMs. The advantages to this system includea greater degree of precision and accuracy associated with multiple datapoints being taken, as well as obviating the need for a referenceelectrode in the system.

Further, in some embodiments, the present invention provides a matrixmaterial (a sol-gel or other polymeric material) that contains one ormore AIMs and/or one or more ASMs in a suitable electrical environmentsuch that the resulting electrode functions as an AIE.

Suitable ASM materials may include, for example and without limitation:pH sensitive ASMs: anthraquinone (AQ), phenanthrenequinone (PAQ),N,N′-diphenyl-p-phenylenediamine (DPPD), anthracene, naphthaquinone,para-benzoquinone, diazo-containing compounds, porphyrins,nicotinamides, including NADH, NAD⁺ and N-methylnicotinamide, quinonethiol, monoquaternized N-alkyl-4,4′-bipyridinium, RuO, and Ni(OH)₂, andderivatives of those compounds; CO-sensitive ASMs: ferrocenylferraazetine disulfide; alkaline metal cation sensitive ASMs:1,1′-(1,4,10,13-tetraoxa-7,1-diazacyclooctadecane-7,16-diyl dimethyl),ferrocenyl thiol, other ferrocene derivatives containing covalentlyattached cryptands, and certain metal complexes with Fe²⁺/Fe3⁺,Co2⁺/Co3⁺, Cu+/Cu2⁺. Suitable ASMs are described, for example, inHammond, et al., J. Chem. Soc. Perkin. Trans. 707 (1983); Medina et al.,J. Chem. Soc. Chem. Commun. 290 (1991); Shu and Wrighton, J. Phys. Chem.92, 5221 (1988), and PCT application no. PCT/US 10/28726. Illustrativeexamples include the above ferrocenyl ferraazetine and ferrocenylcryptands, in which an ordinarily chemically insensitive redox center(ferrocene) is covalently linked to a chemical recognition site in sucha way as to make its redox potential chemically sensitive. Also suitableare molecules or polymers in which the sensor and referencefunctionalities are covalently linked such as1-hydro-1′-(6(pyrrol-1-yl)hexyl-4,4′-bipyridinium bis(hexafluoro-phosphate), as described by Shu and Wrighton, J. Phys. Chem.92, 5221 (1988).

In some embodiments, the WE comprises two or more ASMs sensitive to thesame analyte species, which are selected so as to provide a moresensitive measurement than is provided by a single ASM while minimizingthe possibility of introducing additional overlapping peaks which mustbe resolved to determine analyte concentration. In some examples of thisembodiment, the WE comprises both phenanthrenequinone (PAQ) andanthraquinone (AQ). In other embodiments, the WE comprises two or moreASMs sensitive to the same analyte species. Such ASMs could be selectedto ensure that not all of them are equally susceptible to potentiallyinterfering species that may be present in a test sample. Compounds suchas benzalkonium chloride, a quaternary ammonium chloride, may adverselyinterfere with ASMs such as AQ and PAQ, for example. An ASM lesssusceptible to such interference may be for example a derivative withone or more functional groups that sterically or ionically hinder theapproach of an interfering species. Alternatively, an ASM can becovalently attached to a matrix material that provides such hindrance.

In other aspects, the WE may further comprise an AIM as an internalstandard, as described above. Still further, in some embodiments the WEcomprises two or more ASMs, each ASM being selected for sensitivity to adifferent analyte species.

In one aspect, the present invention provides a matrix material andcorresponding electrodes, probes, pH meters and other analyte sensingdevices in which an ASM (and/or AIM) is covalently attached to a matrixmaterial prepared from a silane-modified ASM (and/or AIM) precursor andalkoxysilanes using a sol-gel process. Sol-gel processing is a techniqueoften used in materials science that transforms a colloidal solution(sol) to an integrated network matrix material (gel). The resultingmatrix material exhibits certain characteristics of ceramic and glass,as well as the pH-responsive functionality (in the case where the redoxactive material is an ASM selected for use in a WE to be used in a pHsensor) required in a pH sensor. Preparation of these matrices caninvolve the following illustrative compounds, compositions, and methodsof the invention below.

In a first step, a silane precursor with the general structure ofFormula (I), below, or salts, hydrates and/or solvates thereof, issynthesized:

wherein: at least two and typically all three R₁, R₂ and R₃ areindependently alkoxy or aryloxy, and if only two are alkoxy or aryloxythe third may be alkyl or aryl; X₁ is —O— or a chemical bond; L is alinker; Y₁ is —CO₂H, halogen, —OH, —NHR₄, —SO₂H, —R₅CO, —P(O)(OR₆)(OH),—N₃ or —CN; R₄, R₅ and R₆ are independently hydrogen, alkyl, or aryl.The composition of the silane-modified ASM precursor, including thenature of the -L- linkage, determines the electrochemical response,longevity, and chemical resistance of the resulting electrode and sensorcontaining the electrode.

Generally, X₁ can be any kind of chemical functionality that can form acovalent bond with silicon, and many such functionalities are known tothose of skill in the art. In some embodiments, X₁ is simply a chemicalbond. Connected to X₁ is a linking moiety of the formula Y₁-L, where Lis a linker and Y₁ is a linking group. The nature of linker L andlinking group Y₁ can, as will be appreciated by those of skill in theart upon contemplation of this disclosure, vary extensively. The linkerL may be hydrophilic or hydrophobic, long or short, rigid, semirigid orflexible.

A wide variety of linkers L comprised of stable bonds suitable forspacing linking groups such as Y₁ from the silicon group are known inthe art, and include, by way of example and not limitation, alkyl, aryl,arylalkyl, polycyclic aryls, esters, ethers, polymeric ethers and thelike. Thus, linker L may include single, double, triple or aromaticcarbon-carbon bonds, etc. Further, alternative embodiments of L includebranched structures that influence the spatial configuration of the ASM,including orientation, distance from the sol gel network, theflexibility of the linkage, and/or electron transfer efficiency. In someembodiments, L is a conjugated system or multiple conjugated systems.

Choosing a suitable linker will be within the capabilities of thosehaving skill in the art upon contemplation of this disclosure. Forexample, where a rigid linker is desired, L can be a rigidpolyunsaturated alkyl or an aryl, biaryl, etc. Where a flexible linkeris desired, L can be a flexible saturated alkanyl. Hydrophilic linkerscan be, for example, polyols (polyalcohols), such as poly(vinyl alcohol)and its derivatives, or polyethers, such as polyalkyleneglycols.Hydrophobic linkers can be, for example, alkyls or aryls.

Alternative embodiments of L include alkyl, aryl, allyl, ether, estersalkoxyl, amide, sulfonamide, and other linkages, including heterocyclic,linear, cyclic, acyclic, or mixed conjugated linkages. The linking groupY₁ should be capable of mediating formation of a covalent bond with acomplementary reactive functionality of an ASM to provide an isolatedsilane-modified ASM precursor of the invention. Accordingly, linkinggroup Y₁ can be any reactive functional group known to be suitable forsuch purposes by those of skill in the art upon contemplation of thisdisclosure. Y₁ can be for example, a photochemically activated group, anelectrochemically activated group, a free radical donor, a free radicalacceptor, a nucleophilic group or an electrophilic group. However, thoseof skill in the art will recognize that a variety of functional groupsthat are typically unreactive under certain reaction conditions can beactivated to become reactive. Groups that can be activated to becomereactive include, e.g., alcohols, carboxylic acids, including saltsthereof.

Thus, in some embodiments, Y₁ is —CO₂H, halogen, —OH, —NHR₄, —SO₂H,—R₅CO, —P(O)(OR₆)(OH), —N₃ or —CN. In other embodiments, Y₁ is —CO₂H,halogen, —OH, —NHR₄ or —N₃

Some embodiments of Y₁-L include for example, compounds where L is—(CH₂)_(n)—, n is an integer between 1 and 8, Y₁ is CO₂H, halogen, —OH,—NHR₄, —SO₂H, —R₅CO, —P(O)(OR₆)(OH), —N₃ or —CN. In some embodiments, Y₁is —CO₂H, halogen, —OH, —NHR₄ or —N₃. In some embodiments, L is—(C₂H₂)_(n)— where n is an integer between 1 and 24.

In some embodiments, R₁, R₂ and R₃ are independently alkoxy, L is—(CH₂)_(n), X₁ is a chemical bond, Y₁ is —CO₂H, halogen, —OH, —NHR₄ or—N₃ and n is an integer between 2 and 6. In other embodiments, thesilane precursor has the structure of Formula (II):

In still other embodiments, the silane precursor has the structure ofFormula (III):

A wide variety of conventional methods may be used to prepare the silaneprecursors above and well within the ambit of the skilled artisan. Forexample, nucleophilic displacement of a silyl chloride (i.e.,Cl—SiR₁R₂R₃) with Y₁L-M or Y₁-L-O-M where M is a metal may provide thesilane precursors above.

In a second step, an ASM (or AIM) group is attached to the silaneprecursor to provide an ASM (or AIM) silane precursor of the inventionof Formula (IV) depicted below (ASM1 is also represented herein asASM₁):

wherein: at least two and typically all three R₁, R₂ and R₃ areindependently alkoxy or aryloxy, and if only two are alkoxy or aryloxythe third may be alkyl or aryl; X₁ is —O— or a chemical bond; L is alinker; Y₁′ is —CO₂NR₄—, —O—, —NR₄CO—, —SO₂—, —R₅CO—, —P(O)(OR₆)O—,—CO₂—, —O₂C—, —NR₄O₂—, —O₂CNR₄, —N═N— or a chemical bond; ASM₁ is ananalyte sensitive material (or analyte insensitive material, or aderivative of either); and R₄, R₅ and R₆ are independently hydrogen,alkyl, or aryl. In general, ASM₁ is considered a derivative of an ASM(or AIM), because, in the structure of Formula (IV), it differs from thecorresponding ASM (or AIM) by loss of a hydrogen atom as necessitated byformation of a covalent bond to either Y₁ or L. In some embodiments, R₁,R₂ and R₃ are independently alkoxy; L is —(CH₂)_(n), X₁ is a chemicalbond; Y₁′ is —CO₂NH, or —O—, or —NHR₄; and n is an integer between 2 and6.

In some embodiments, ASM₁ is selected from the group consisting ofFormulas (V), (VI), (VII) or (VIII):

In other embodiments, ASM₁ is derived from an ASM selected from a groupconsisting of Formulas (IX), (X), (XI), (XII), (XIII), (XIV), (XV) and(XVI):

wherein R₂₀, R₂₁, R₂₂, R₂₃, R₂₄, R₂₅, R₂₆, R₂₇, R₂₈, R₂₉, R₃₀, R₃₁, R₃₂and R₃₃ are independently hydrogen, —CO₂H, halogen, —OH, —NHR₄, —SO₂H,—R₅CO, —P(O)(OR₆)(OH), —N₃, —CN, alkyl, aryl or alkoxy with the provisothat least one substituent is —CO₂H, halogen, —OH, —NHR₄, —SO₂H, —R₅CO,—P(O)(OR₆)(OH), N₃ or —CN.

In some embodiments, the ASM silane precursor of the invention has thestructure of Formula (XVII):

For clarity, the CON in the preceding structure is a carbonyl grouplinked to a N (the N thus has a hydrogen attached to it that is notshown in the structure). In other embodiments, the ASM silane precursorof the invention has the structure of Formula (XVIII):

Still, in other embodiments, the ASM silane precursor of the inventionhas the structure of Formula (XIX):

Generally, the ASM silane precursor of Formula (IV) can be assembledfrom the silane precursor of Formula (I) and an appropriatelyfunctionalized ASM using conventional methods of organic synthesis.These include, for example, ester, amide, and sulfonamide condensations,alkylations, 1-3 dipolar cycloadditions, and carbene, nitrene and freeradical additions between complementarily functionalized compounds ofFormula (I) and ASMs. Thus, the invention also provides new compoundsthat are complementarily functionalized ASMs suitable for use in suchsynthetic methods.

In a third step a sol comprising the ASM precursor, a polyalkylorthosilicate or similar multifunctional silane, solvents, an acidcatalyst, and optionally other additives is prepared. At this point, thesilane groups in the ASM precursor and the polyalkyl orthosilicate orsimilar multifunctional silane hydrolyze and undergo crosslinking,converting sol to gel. The resultant sol-gel is heterogeneous to somedegree, comprising both liquid and solid regions whose morphologies canrange from discrete particles to continuous structures of varyingporosities. In the process, the ASM is also incorporated into thecrosslinked network to form a matrix of the invention, as shown in thefollowing reaction scheme:

The term “ormosil” stands for “organically modified silica/silicate.” Inthe scheme above, at least two and typically all three R₁, R₂ and R₃ areindependently alkoxy or aryloxy, and if only two are alkoxy or aryloxy,the third may be alkyl or aryl. Each of these moieties is independentlyselectable. Thus, if R₁ in Formula (IV) is methoxy, R₁ in Formula (XX)can be aryloxy or another alkox, and if R₂ and R₃ in Formula (XX) arealkoxy or aryloxy, then R₁ in Formula (XX) can be alkyl or aryl, even ifR₁ in Formula (IV) is methoxy. Typically, in compounds of Formula I,Formula (IV), and Formula (XX), at least two and often all three of R₁,R₂ and R₃ are independently methoxy or ethoxy. Following formation ofthe sol-gel, a coating of this material, which is still in liquid form,is applied onto a prepared conductive substrate. Finally, solvents areremoved under controlled thermal treatment conditions which results in astable structure with ASM covalently bonded to, and dispersedthroughout, the matrix material which is substantially in contact withthe conductive substrate, thereby forming an electrode of the invention.Thus, the matrix is the analyte (e.g. pH)-sensitive surface of a workingelectrode of the invention, or is itself formed into the electrode (inthose embodiments where no conductive substrate is employed, but thematrix material itself is conducting). If an AIM is used as ASM1, thenthe matrix serves as the active surface of the AIE, or is itself formedinto the electrode (in those embodiments where no conductive substrateis employed, but the matrix material itself is conducting).

Those skilled in the art will recognize that this method may be used tocreate sensors containing one or more ASMs, one or more AIMs, andcombinations of ASM(s) and/or AIM(s). Such combinations can provideadditional response signals that vary with analyte identity andconcentration, and/or reference signals substantially unchanged withanalyte identity and concentration. Such combinations are thereforewithin the scope of the present invention.

In some embodiments, the sol-gel matrix material comprises a single ASM(or a single AIM). For example, some embodiments of single ASM pH WEscomprise a sol-gel matrix material in which the ASM is selected from thegroup of compounds consisting of 2-carboxy AQ, 2-N-BOCethylene diamineAQ, 5,12-naphthacene quinone, 1-acetyl amido AQ, 2-carboxamido AQ, and3-carboxamido PAQ. In other embodiments, a single ASM WE comprises asol-gel matrix material in which the ASM is 2-(beta-naphthol)methylanthraquinone.

In other embodiments, the sol-gel matrix material of the presentinvention comprises at least two or more ASM (or ASM and AIM) compounds.

Some embodiments of the present invention comprise a sol-gel matrixmaterial having at least one ASM and optionally comprise one or moreadditional ASMs and/or an AIM(s). Both the ASMs and AIMs includeredox-active materials exhibiting reversible redox activity withwell-defined cyclic voltammetry methods.

Some embodiments of the present invention may further include a sol-gelmatrix material incorporating an AIM component having a redox potentialthat is substantially insensitive to the chemical medium to which thesensor is introduced. Such AIMs may include, for example and withoutlimitation AIMs selected from the group comprising ferrocene, n-butylferrocene, K₄Fe(CN)₆, polyvinyl ferrocene, nickel hexacyanoferrate,ferrocene polymers and copolymers, including ferrocene styrene copolymerand ferrocene styrene cross-linked copolymer, nickel cyclam, and others.Further, non-limiting examples include ferrocenyl thiol,polyvinyl-ferrocene, viologen, polyviologen and polythiophene. Otherembodiments include AIMs comprising ordinarily chemically sensitivematerials which are chemically isolated, yet in electrical contact withthe chemical medium or analyte sample.

In another aspect, an AIM, such as, for example, a substituted ferrocenesuch as that depicted below as Formula (XXI), is covalently attached toa matrix material prepared from a silane-modified AIM precursor andalkoxysilanes using a sol-gel process.

This AIM precursor, and similar AIM precursors featuring alternativelinking groups, are compounds of the invention that can be used as thebasis of an AIM covalently attached to a matrix material based on thesol-gel chemistry-based methods of the invention. The AIM precursor canbe used in admixture with an ASM precursor to form an electrode thatproduces an analyte-sensitive signal and an analyte-insensitive signal.Alternatively the AIM precursor can be used to form a discrete structure(an AIE) generating a single, analyte-insensitive signal, which can beused in conjunction with an analyte-sensitive signal produced by aco-located WE. In another aspect of the invention, the ASM is covalentlyattached to a matrix material prepared from a synthetic polymer. Theresulting matrix material exhibits both the intrinsic properties of thepolymer, which functions as a framework to secure the ASM while offeringspatial dispersion for contact between analyte and the ASM, bonding witha conductive substrate, and the analyte (e.g. pH)-responsivefunctionality required in a WE or the appropriate functionality requiredfor an AIE. Synthetic polymer attributes of interest as ASM matrixmaterials include, but are not limited to, good film-forming properties,compatibility with conductive substrates, adjustable mechanicalproperties such as rigidity, strength, ability to be shaped intodifferent form factors, and a wide range of properties attainable byblending, copolymerization, crosslinking, grafting, and physical orchemical modifications at the bulk or surface levels. These attributespermit the design and fabrication of electrodes in sizes, form factors,and performance to meet different requirements. A general approach insome methods of the invention is to use an ASM with suitable functionalgroups reactive to complementary functional groups in the matrixpolymer. Those skilled in the art will recognize, upon contemplation ofthis disclosure, that the ASM can be situated along the backbone of thematrix polymer and/or on branches or side-chains of the matrix polymer,and that by introducing the ASM as functionalized tethers, crosslinkers,or chain-extenders, a myriad of different matrices of the invention canbe prepared.

In yet another aspect, an ASM is incorporated into inherently conductivepolymers to derive sensors in which the electrochemical signalsgenerated by the ASM can be efficiently captured and transmitteddirectly to electronic processing circuits via the conductive polymer.In this aspect, the resulting electrode does not have a traditionalconductive substrate. Eliminating the interface between the pH-sensitivematrix material and a separate conductive substrate resolves severalproblems associated with the physical, chemical, and electroniccompatibility of dissimilar materials. The use of polymers, includingconductive polymers, helps reduce the complexity and improve designflexibility and manufacturability of electrodes assuming various formfactors, and so provides means to fabricate the flexible sensors andminiaturized sensors of the invention.

In some embodiments, a working electrode is prepared using matrixmaterial of the invention based on AQ covalently bound to poly(vinylalcohol), or PVA. The finished structure comprises an olefinic backboneand an ether-linked side chain to which the AQ moiety is tethered. PVAexhibits excellent resistance to chemical attack, as does the etherlinkage connecting the AQ. PVA is also an excellent film-formingpolymer. Furthermore, PVA can be crosslinked by chemical and thermalmeans, further enhancing the dimensional and environmental stability ofthe finished structure. These are all desirable properties in a pH (orother analyte) sensor expected to encounter a wide range of operatingconditions.

In other embodiments, polymers exhibiting good physical, chemical, andmechanical attributes that can be functionalized to attach ASM (or AIM)moieties are generally viable alternatives PVA, and preferredalternatives to PVA for specific applications and/or fabricationmethods. Such polymers include, but are not limited to, derivatives ofpolysulfone, polyethersulfone, polyamides, polysulfonamides, polyimides,polyesters, vinyl polymers, polyphenylene sulfide, polysaccharides,cellulose, derivatives thereof, copolymers thereof, blends thereof, andcomposites thereof. ASM (or AIM) attachment methods include, but are notlimited to, reaction with functionalized ASMs (AIMs), grafting ofpolymers already containing ASMs (or AIMs) as tethers, interpenetratingnetworks of multiple polymer or oligomer components in which at leastone of the components comprise covalently-attached ASM (or AIM)moieties.

In other aspects of this invention, a combination of a single ASM, aplurality of ASMs, an AIM, and/or a plurality of AIMs, are covalentlyattached to a matrix material prepared from alkoxysilanes and asilane-modified ASM (or AIM) precursor using a sol-gel process, orcovalently attached to a matrix material prepared from an optionallycrosslinked polymer.

In some embodiments, the concentration of ASM on the WE (or AIM on theWE or the AIE) is increased by applying multiple layers of ASM (orAIM)-containing sol-gel matrix material or polymeric matrix material tothe WE (or AIE) substrate. In accordance with the present invention, theordinarily skilled artisan can control the amount of pure ASM (or AIM)entrapped within the matrix material onto the substrate, therebypermitting the manufacture of WEs (or AIEs) and probes containing themhaving a size and shape appropriate to a given application to achieveadditional benefits of the invention.

In some embodiments, the WE comprises ASM present in a sufficient amountto result in a pH-dependent signal of between 2 and 300 microamperes. Insome embodiments, the size and shape of the WE are chosen so as tominimize deleterious electrochemical effects among the WE, RE and CE (orWE, AIE, and CE) while maintaining WE performance sufficient to allow auser to distinguish the analyte-dependent signal over background noisewhile maintaining signal quality.

The WEs (and AIEs) of the present invention may be configured so as tobe removable from the probe, allowing them to be easily interchanged orreplaced according to the required design and functionality. The WEs ofthe invention can be configured and programmed to replace a traditionalglass probe in a traditional pH meter and/or to generate a signal thatis transmitted by electrical wiring, or via electromagnetic means notrequiring wires, to a readout device (see U.S. provisional patentapplication Serial Nos. 61/475,164, filed 13 Apr. 2011, and 61/475,590,filed 14 Apr. 2011, both of which are incorporated herein by reference).

Some embodiments of the present invention further provide improvedanalyte sensors having one or more WEs, each comprising one or more ASMsentrapped within a sol-gel or other polymeric matrix material anddisposed on a substrate and in electrical connection with thatsubstrate.

In some aspects, the WE comprises an AQ derivative as an ASM. In otheraspects, the present invention provides a WE that comprisesphenanthrenequinone (PAQ) or a derivative thereof as an ASM. Further, inother aspects the present invention provides a WE that comprisesortho-benzoquinone (OQ) or a derivative thereof as an ASM. Stillfurther, in other aspects, the present invention provides a WE thatcomprises N,N-diphenyl para-phenylene diamine (DPPD) or a derivativethereof as an ASM.

In some aspects, the present invention provides an electrode thatcomprises anthracene (AC) or a derivative thereof as an ASM. In otheraspects, the present invention provides an electrode that comprisesnaphthoquinone (NQ) or a derivative thereof as an ASM. Further, in otheraspects the present invention comprises provides an electrode thatpara-benzoquinone (PQ) or a derivative thereof as an ASM.

Those of skill in the art will appreciate, in view of this disclosurethat, in general, many of the teachings herein that concern ASMs areequally applicable to AIMs.

A variety of substrate materials are suitable for use in the WEs andAIEs of the present invention. These include but are not limited tocarbon, carbon allotropes, and derivatives thereof, various carbon-basedmaterials, transition metals, noble metals such as gold and platinum,conductive metal alloys, various conductive polymers and copolymers andcompounds and derivatives thereof, polymer blends and polymercomposites, semiconductive materials such as silicon and derivativesthereof, including doped silicon and doped semiconductive materials, andadditional suitable materials known to those of skill in the art.

In some aspects, the substrate is or comprises carbon. A variety ofcarbon substrates are suitable for use as substrate material in theelectrodes of the present invention, including but not limited to carbonallotropes such as pyrolytic graphite, graphite, amorphous carbon,carbon black, single- or multi-walled carbon nanotubes, graphene, glassycarbon, boron-doped diamond, pyrolyzed photoresist films, and othersknown in the art. Additionally, all of the above carbon allotropes maybe dispersed in powder form in a suitable binder, or formed in-situ onthe substrate surface. Such binders include organic or inorganicpolymers, and adhesive materials. In some embodiments, the substrate isgraphite powder and the binder is epoxy resin. In other embodiments, thesubstrate is a graphite rod. In other embodiments, the substrate is acarbon fiber composite. In other embodiments, the substrate is agraphite-filled polymer exemplified by, but not limited to,polyphenylene sulfide. In other embodiments, the substrate comprises asurface coating of an ink formulated with one or more carbon allotropes.

Thus, in some embodiments, the substrate comprises a composite materialcomprising graphite and a binder, such as an epoxy. In some embodiments,the substrate comprises a composite material comprising carbon fibersand one or more binders. In some embodiments, the substrate comprises aconductive polymer such as polyaniline, polypyrrole, or various carbonallotrope-filled polymers, where the polymer components may include,without limitation, polyphenylene sulfide, polyolefins, polyamides,polyimides, polyesters, polysulfone, polyethersulfone, various vinylpolymers, cellulose, poly(amino acids), derivatives thereof, copolymersthereof, blends thereof, and composites thereof. In some embodiments,the substrate comprises materials surface-treated by corona discharge,electron beam, gamma irradiation, plasma, and other forms of irradiationthat result in an activated surface by ion or free radical generation.Optionally, such activated surface may be further modified to enableattachment of ASM (or AIM) derivatives or matrix materials containingcovalently attached ASM (or AIM) moieties.

In some aspects of the invention, the surface of the substrate iscleaned or otherwise “prepared” prior to applying the ASM (or AIM)containing sol-gel or polymeric matrix material. Suitable methods forgraphite/epoxy substrates and other substrates include but are notlimited to sanding and/or polishing the surface of the substrate, whichmay be formed into a plug, followed by directing a stream of pressurizedair or other gas onto the substrate surface, and optionally sonicatingin a suitable solvent, to dislodge particulates resulting from sandingor polishing. Alternatively, and optionally, the substrate surface maybe cleaned with various solvents, alkalis, and/or acids, followed bythorough removal of such cleaning agents by means well known to theskilled practitioner. These methods, applied selectively to substratescompatible with the treatment conditions and chemicals, serve to removesuperficial contamination prior to attachment of the ASM (orAIM)-containing matrix material. The resulting WE (or AIE) is capable ofrendering an analyte-dependent signal (or appropriate AIE signal) havingsuperior useful lifetime as compared to the signals produced by WEs (orAIEs) in which the ASM (or AIM) is physically adsorbed without employinga matrix material or is covalently attached to the substrate.

A substrate acts as a self-contained entity that serves as a physicaland electrical bridging unit between one or more ASMs (or AIMs) withinthe sol-gel or polymeric matrix material, and an electrical conduit,such as a wire. The physical function of the substrate is to provide asupport for the ASM (or AIM) sol-gel or polymeric matrix material suchthat the electrode may be brought into direct contact with a sample ofinterest, typically a liquid, safely and conveniently in the form of aprobe or a functionally equivalent assembly. The substrate and matrixmaterial thereby allow the ASMs (or AIMs) to interact with the sample ofinterest. The electrical function of the substrate is to propagatecharge carriers such as electrons from the electrical conduit to the ASM(or AIM) to enable a redox reaction. In some embodiments, a substratematerial is selected to be chemically inert to the anticipatedenvironments of the sample to be analyzed, and an ability to conductelectrical current with minimal loss.

As discussed above, in some embodiments, the pH or other analyte probeof the invention further includes a reference electrode (RE). A numberof reference electrodes suitable for use in a probe of the presentinvention are known in the art. See, for example, Bard and Faulkner,“Electrochemical Methods: Fundamentals and Applications” (Wiley 2001),incorporated herein by reference.

In some embodiments of the invention, the reference electrode comprisesa chloridized silver wire surrounded by an electrolytic solution, asdescribed in the examples below. In other embodiments, the RE comprisesonly a chloridized silver wire. In other embodiments, the RE comprisesan iodide/tri-iodide system as described in U.S. Pat. No. 4,495,050,incorporated herein by reference.

Optionally, “pseudo-reference” (PRE) (sometimes referred to as“quasi-reference”) electrodes are used, particularly in non-aqueouselectrolytes, in an analyte sensor of the invention. An illustrative butnon-limiting example of a PRE is a silver wire, commonly used innon-aqueous electrochemistry; other PREs may be used according to theparticular application.

In some embodiments, the surface area of the RE exposed to the analytesample is selected so as to minimize or eliminate intra-electrodeelectrochemical effects that adversely affect analyte-dependent signalquality.

The present invention also provides a variety of embodiments in which asolid-state working electrode (WE) featuring a redox-activeanalyte-sensitive material is operated in conjunction with aconventional RE in the same pH metering system. This hybrid approachcombines the robustness inherent in solid-state devices and the acceptedreference standard upon which much of electrochemistry science is based.

Thus, one of ordinary skill in the art will appreciate the unique hybridconfiguration of various embodiments of the present invention. Inparticular, one of ordinary skill in the art will appreciate the presentcombination of a solid-state WE with a conventional RE, typically with aCE as well. This hybrid configuration provides a pH probe assemblyhaving the reliability of a conventional RE without the unwanted andcumbersome calibration requirements of traditional glass workingelectrode. Thus, the present invention provides new and usefulcombinations of electrodes that overcome the limitations of traditionalpH (or other analyte) probes and metering systems.

In some embodiments the RE is modified to include a miniature referenceassembly configured in the shape of the letter “J”, as described in PCTPat. Pub. No. 2010/111531, incorporated herein by reference. Theminiature reference electrode is alternatively placed at the proximalend of a traditional RE such that the chloridized silver wire of theminiature reference assembly is positioned adjacent or proximal to theWE of the pH probe. The shape of the miniature reference ensures that atleast a portion of the chloridized silver wire is located in closeproximity to the sample solution. Thus, the temperature of the wire issubstantially similar to the WE, typically within 10% of the temperatureof the WE and the analyte solution. In some embodiments, the temperatureof the wire varies less than 2 degrees from the temperature of the WEfollowing equilibration of the probe assembly in the analyte-containingsample. In other embodiments, the temperature of the wire varies lessthan 1 degree from the temperature of the WE following equilibration ofthe system. Still further, in some embodiments the temperature of thewire varies less than 3 degrees from the temperature of the WE followingequilibration. Subjecting the silver wire to substantially the sametemperature as the WE and/or sample solution reduces the temperaturesensitivity of the sensor thereby increasing the accuracy of the pHprobe and meter.

Some embodiments of the present invention are probes that include acounter electrode (CE). In operation, the CE serves as an electronsource or sink, thereby delivering current to the sample and allowing itto flow through the pH probe system.

Suitable CEs are known in the art. See, for example, Bard and Faulkner,above. To avoid unwanted electrochemical redox processes occurring atthe CE that can interfere with the signal measured at the WE, the CE istypically made of a relatively chemically inert material, commonlystainless steel, carbon (e.g., graphite) or platinum.

In some embodiments, as illustrated in the examples below, the CE is agraphite or carbon-based rod. In other embodiments the CE is acarbon-fiber tube. Still further, in some embodiments the CE is anelectrically conductive material, as known in the art.

Various references describe the importance of the WE:CE surface arearatio to sensor performance. In various embodiments of the presentinvention, the surface area of the CE exposed to the analyte sample isselected so as to minimize or eliminate intra-electrode electrochemicaleffects that adversely affect analyte-dependent signal quality andlongevity, as described herein. In some embodiments, the ratio of thesurface area of the CE to that of the WE is from about 1:1 to about1:10. In other embodiments this ratio is about 1:2, and in furtherembodiments the ratio is about 1:1.5.

In some embodiments, the CE comprises an electrically conductivecarbon-fiber tube having a hollow inner lumen for housing various othercomponents of the pH sensor. The carbon-fiber tube is electricallycoupled to a preamplifier module whereby a voltage is applied to thesample solution via the CE. One skilled in the art will appreciate that,in addition to providing electrochemical cell driving potential, theelectrically conductive, low-impedance CE serves as an electromagneticshield to protect components housed within CE, especially thehigh-impedance RE, from external electromagnetic interference. In someembodiments, a coaxial configuration is implemented whereby an externalposition of the CE provides electromagnetic shielding to the RE and WE,which electrodes are concentrically or approximately concentricallypositioned within the CE. One skilled in the art will further appreciatethat the shielding function of the CE is not dependent upon theconcentric positioning of the RE and WE. Rather, one skilled in the artwill appreciate that the exact positions of the RE and WE may beinternally altered relative to the external position of the CE and stillreceive the shielding protection as discussed above.

In some embodiments, the invention provides an analyte insensitiveelectrode (AIE) that is used in lieu of a conventional RE.

The AIE is capable of generating a substantially analyte insensitivesignal in response to the application of an electrical stimulus appliedto the sample being analyzed in the course of making voltammetric oramperometric measurements of analyte concentration in the sample. TheAIE provides a predictable signal useful as an internal standard (inother words, a standard internal to the system) with which ananalyte-sensitive signal may be continuously compared, and thereforepermit greater accuracy and reproducibility in determining analyteconcentration.

Thus, in some embodiments of the present invention, an AIE is used inthe electrochemical analyte sensing device to generate a substantiallyanalyte-insensitive electrical response when an electrical stimulus isapplied to an analyte sample in the course of making voltammetric and/oramperometric measurements of analyte concentration.

The teachings of the current invention regarding different WEchemistries are also applicable to certain embodiments of the AIE.Specifically, an AIE can feature the same pH- (or other analyte-)responsive surface chemistry as the WE and, due to itsspecially-formulated constant chemical environment and the PRE itcontains, used to replace the conventional RE (such as Ag|AgCl|KCl). SeePCT Pat. Pub. No. 2010/104962, incorporated herein by reference.

Conventional REs operate by establishing a stable, well-characterizedelectrode potential. The stability of this electrode potential derivesfrom a redox system with constant activities of each participant of theredox reaction. Stable electrode potentials are obtainable usingelectrodes with covalently-attached ASM (or AIM) matrix material as thesensing surface. These electrodes generate highly reproducible electrodepotentials in a constant chemical environment such as that provided by abuffer solution. Thus, one embodiment of an AIE of the inventioncontains a redox-active matrix material of the invention, optionallyattached to a substrate, and a PRE in a buffer solution contained withinan enclosed volume. This enclosed volume is, in turn, in fluid andelectrical communication through a liquid barrier such as a porous fritwith the analyte solution. In operation, this AIE is co-located with theWE and the CE, and each electrode is in direct contact with the analytesolution. Regardless of whether a redox active material is characterizedas an ASM or AIM, it can be made analyte-insensitive by sequestration ina properly formulated ionic medium, as contained in an AIE.

In operation, the AIE exhibits an electrode potential dependent largelyon the nature of the redox active material and the nature of theconstant chemical environment in which it is sequestered, i.e., in aproperly formulated ionic medium (see U.S. provisional patentapplication No. 61/434,800, filed 20 Jan. 11). Provided that anappropriate liquid barrier is selected, convective mixing between theanalyte sample and a properly formulated internal buffer solution can bereduced to insignificant levels on the time scale of interest. Anyintrusion of the analyte sample across the liquid barrier will onlyexert a minimal effect on the chemical environment because of theinherent ability of the buffer solution to mitigate pH shifts due tocomposition changes. The result is an exceptionally stable electrodepotential compared with conventional REs such as Ag|AgCl|KCl, in whichthe KCl solution has no significant buffering capacity. This fundamentaladvantage of the AIE over conventional REs, coupled with the stabilityderived from covalently-attached ASM (or AIM) matrix material, resultsin unexpectedly superior performance of this new class of pH probescontaining such electrodes.

The AIE and the WE function similarly in that both are redox-activeelectrodes. In one embodiment of a sensor of the invention, theredox-active material and matrix containing it in the AIE and the WE areidentical or substantially similar. In another embodiment of the sensor,the AIE and the WE have different chemical compositions, i.e., theydiffer in the redox-active compound employed or in the matrix employed,or both. The latter embodiments offer additional degrees of freedom andgreatly expand the different types of pH (and other analyte) sensingsystems of the invention, in that the AIE and the WE may be individuallytailored to deliver the most beneficial combination of physical andperformance attributes. For example, the WE can be based on a chemicalcomposition designed to offer the highest accuracy over a broad pHrange, whereas an alternate WE can be based on a chemical compositiondesigned to withstand aggressive chemical environments such as strongacids or alkalis. In either case, the AIE can be based on a chemicalcomposition exhibiting the highest precision in the presence of aneutral buffer to offer the longest life expectancy for the AIE. Thechemistry options described in the present invention, including variouslinking chemistries, enable control of WE properties to meet thesediverse needs.

In various embodiments of the current invention, an AIE featuringconstant chemical environments in the form of a semi-solid or a solidare employed. See U.S. provisional patent application No. 61/434,800,filed 20 Jan. 11, incorporated herein by reference. Thus, in oneembodiment, the reference solution of the AIE is replaced with a solid(the “reference material”) that provides buffering capacity, electricalconductivity, and ionic permeability. The reference material is ahydrophilic solid with sufficient ionic content to serve as a conduit ofhydrogen ions in the analyte solution, and a conductor of electricalcurrent between the CE and the AIE. The reference material is in directcontact with the analyte on one side, and with the functional surface ofthe redox-active material and with the PRE on the other side. In oneembodiment, the reference material is selectively permeable to hydrogenions but substantially impermeable to other entities in the analytesolution, so that the redox-active material within the AIE will not beexposed to a changing chemical environment during use. In anotherembodiment, an additional liquid barrier is used at the interfacebetween the reference material and the analyte. Such a barrier may be inthe form of an ionic liquid or room-temperature ionic liquid. In thisway, the AIE overcomes problems associated with maintaining wetreference systems, may be stored dry over long periods until use, andmay be kept dry between measurements in analyte solutions, i.e., haswet-dry reversibility.

Suitable reference materials include crosslinked ionic polymers withcompositions that mimic those of liquid buffer solutions; for example, acombination of strongly basic and weakly acidic functionality, or astrongly acidic and weakly basic functionality.

The principles and methodologies of the current invention regarding thecreation of WEs embodying certain desirable attributes can be applied tovarious designs of electrodes, probes, sensor assemblies, analytemeasuring devices, meters and systems, and instrumentation. Each ofthese deployments will benefit from the advantages of electrodes of thecurrent invention over conventional electrode systems, in particularthose based on glass probes, and specifically glass pH probes.

Thus, in one embodiment of the invention, an amperometric probe isprovided for use in conjunction with conventional pH meters as auniversal replacement of the glass probe. See U.S. provisional patentapplication No. 61/475,500, filed 14 Apr. 11, incorporated herein byreference. The amperometric probe described in that applicationcomprises a WE, a RE, and a CE. In other embodiments, the currentinvention provides a WE, an AIE, and a CE that together replace theprobe of a traditional pH meter (or other analyte sensing device), asdescribed in that application.

In yet another aspect, the present invention provides an amperometricprobe for measuring pH comprising a probe, signal processing algorithm,and circuitry that enable wired or wireless communication of pH,temperature, and other analyte characteristic information to, and to beprocessed, analyzed, or displayed by, computing devices. See U.S.provisional patent application No. 61/475,164, filed 13 Apr. 11,incorporated herein by reference. The methods, electrodes, and electrodecombinations (e.g. WE, RE, and CE; and WE, AIE, and CE) of the presentinvention can be used in amperometric probes as described in thatapplication.

For an electrode and corresponding probe to achieve maximal longevity,the peak intensity (the magnitude of the current at its maximum) of theWE should decrease minimally over time. As shown in FIG. 1, the ASMsol-gel WE displayed less signal loss as compared to a WE where the ASMis physically adsorbed on the substrate. While the electrodes andsensors prepared via sol-gel (or other polymer) deposition can initiallylose some fraction of their initial signal intensity, as illustrated inthe example described in FIG. 1, the signal in this illustrative examplestabilizes subsequently at a level with acceptable signal-to-noiseratio. These results are significant, because a decrease in signal lossresults in increased detectability and signal-to-noise ratio, and alonger functional lifetime for the WE.

FIG. 2 illustrates the signal stability over a 30 day lifetime of a WEprepared using the sol-gel deposition method of the invention. Inparticular, FIG. 2 demonstrates negligible variation of peak positionfor the ASM sol-gel WE over a 30 day period. Typically, a WE producedusing physical adsorption as the mode of ASM/electrode surfaceinteraction provides useable signals only up to about 2 days. For singleuse and limited use applications, a short useful lifetime may beadequate. However, for applications which demand longer usefullifetimes, such electrodes are inadequate. Thus, the ASM sol-gel (orother polymer matrix) WE (and AIE) of the present invention provides asolution to the problem of short electrode lifetime.

Those of skill in the art will appreciate upon contemplation of thisdisclosure that there are many alternative ways of implementing andrealizing the many benefits and advantages afforded by the variousaspects and embodiments present invention. Accordingly, the presentembodiments are to be considered as illustrative and not restrictive,and the invention is not to be limited to the details given herein, butmay be modified within the scope and equivalents of the appended claims.

All publications and patents cited herein are incorporated by referencein their entirety.

The following examples are provided for illustrative purposes only anddo not limit the scope of the invention.

EXAMPLES Example 1 Synthesis of an Amide-Linked AQ Silane

Synthesis of an amide-linked AQ silane was prepared, as shown in thefollowing reaction scheme:

This example and the following Examples 2 and 3 describe compounds andmethods of the invention for making an ASM-amide-linked sol-gel matrixmaterial and corresponding WE of the invention.Anthraquinone-2-carboxylic acid (0.500 g, 1.98 mmol) was suspended indichloromethane (20 mL) in a vial, and then the vial was purged withnitrogen. 0.2 mL of dimethylformamide (DMF) was added dropwise, followedby addition of oxalyl chloride (0.18 mL, 2.2 mmol). The solution wasstirred for 2 h. Then, diethylaminomethylpolystyrene (1.97 g, 6.3 mmolof amine) was added followed by 3-aminopropyltrimethoxysilane (APTOS)(0.37 mL, 2.1 mmol). Stirring was continued for 16 h at ambienttemperature. The resultant liquid was filtered to remove thediethylaminomethylpolystyrene solid. Additional dichloromethane was usedto rinse the retained solid to recover entrained product. Thedichloromethane was removed in a rotary evaporator to obtain an amberoily liquid. Anhydrous alcohol was used to reconstitute the oily liquidto obtain 20 mL of the amide-linked AQ silane solution.

Example 2 Preparation of Amide-Linked AQ Sol-Gel Matrix Material

A preparation of amide-linked AQ sol-gel matrix material was prepared,as shown in the following reaction scheme:

A sol was prepared by combining ethanol (3.5 mL), water (0.250 mL, 14mmol), 0.1M HCl (0.200 mL, ˜11 mmol water), and TEOS (1.00 mL, 4.48mmol) in a 20 mL vial, as shown in the reaction scheme immediatelyabove. The amide-linked AQ silane solution (1.5 mL, 0.01 mmol/mL, 0.015mmol) was added. The resulting solution was heated at 70° C. withstirring. After 1 h, a solution containing 0.135 g ofoctyltrimethylammonium bromide (0.53 mmol) and 6.5 mL ethanol was added,and stirring was continued at ambient temperature for 1 hour.

Example 3 Preparation of Amide-Linked AQ Sol-Gel Working Electrode

To prepare the WE, a carbon fiber composite rod of 0.180 in diameter wasmounted in a polyetheretherketone (PEEK) surround and sanded until thecarbon disk reaches a 0.06 in thickness. These blank substrates areimmersed in the sol containing the amide-linked AQ described in theprevious paragraph for 30 s, removed from the liquid, and heated in a150° C. oven for 1 h.

Samples of these working electrodes were tested using an Autolabpotentiostat (Metrohm Autolab B.V.) with a pH 7 buffer standard at roomtemperature, with the results shown in Table 1.

TABLE 1 Sample Code Peak position (V) Peak current (μA) AA063 −0.487136.4 AA096 −0.4873 25.1 AA065 −0.4871 36.4 AA032 −0.4880 27.3

Example 4 Synthesis of an Ether-Linked AQ Silane

Synthesis of an ether-linked AQ silane was prepared, as shown in thefollowing reaction scheme:

This example and the following Examples 5 and 6 describe compounds andmethods of the invention for making an ASM-ether-linked sol-gel matrixand corresponding WE of the invention. Sodium hydride [60%, 0.096 g] wasslowly added to a clean and dry three neck round bottom flask under anitrogen atmosphere. Then, 100 ml of anhydrous DMF was added to theflask using a syringe. This mixture was stirred for 30 min. 0.5 g[0.00209 moles] of 2-(hydroxy methyl) anthraquinone was added andstirred until completely dissolved. After 3 h of continuous stirring,0.6096 g [1.2 eq, 0.6 ml] of (3-Bromopropyl) trimethoxy silane was addeddrop-wise with nitrogen blanketing and stirring. The reaction wasconducted at room temperature for 4 days, and then stopped by additionof 10 mL of ethyl acetate, followed by stirring for 1 h. The reactionvessel was opened to air, and the reaction mixture was filtered in aBuchner funnel to remove the salt formed, and the liquid wasconcentrated in a rotary evaporator to remove ethyl acetate and DMF toobtain the ether-linked AQ silane stock solution.

Example 5 Preparation of Ether-Linked AQ Sol-Gel Matrix Material

A preparation of ether-linked AQ sol-gel matrix material was prepared,as shown in the following reaction scheme:

A sol-gel matrix was prepared according to the above reaction scheme asfollows: TEOS (1 mL, 5 mmol) was stirred in ethanol (3.5 mL) at 70° C.Water (0.250 mL, 14 mmol) and 0.1M HCl (0.200 mL, ˜11 mmol water) wereadded. Then, 1.5 mL of the ether-linked AQ silane stock solution wasadded. The combined solution was heated at 70° C. and stirred for 1 h.

Example 6 Preparation of Ether-Linked AQ Sol-Gel Working Electrode

To prepare the WE, a carbon fiber composite rod of 0.180 in diameter wasmounted in a PEEK surround and sanded until the carbon disk reaches a0.06 in thickness. These blank substrates were immersed in the solcontaining the ether-linked AQ described in the previous paragraph for30 s, removed from the liquid, and heated in a 150° C. oven for 1 h.Samples of these working electrodes were tested on an Autolabpotentiostat with a pH 7 buffer standard at room temperature, with theresults shown in Table 2.

TABLE 2 Sample Code Peak position (V) Peak current (μA) AA014 −0.50316.6 AA020 −0.505 16.1 AA042 −0.501 19.5 AA055 −0.505 24.4

Example 7 Synthesis of an Ether-Linked Ferrocene Silane

This example and the following Examples 8 and 9 describe compounds andmethods of the invention for making an AIM-amide-linked sol-gel matrixand corresponding AIE of the invention. Hydroxymethyl ferrocene wasreacted with (3-bromopropyl) trimethoxysilane to obtain the silaneprecursor, as shown in the following reaction scheme:

Example 8 Preparation of Ether-Linked Ferrocene Sol-Gel Matrix Material

A preparation of ether-lined ferrocene sol-gel matrix material wasprepared, as shown in the following reaction scheme:

Example 9 Preparation of Ether-Linked Ferrocene Sol-GelAnalyte-Insensitive Electrode for Replacement of a Conventional RE

The ferrocene (or, more generally, AIM) sol-gel matrix material obtainedas described above may be used in the form of a discrete referenceelectrode, or it may be used in admixture with the ASM sol-gel matrixmaterial described, for example, in Examples 2 or 5. In either case, theAIM and ASM components generate a pH-dependent signal and apH-independent signal, thus providing a means of internal calibration ofthe pH response of the sensor.

Example 10 Synthesis of AQ-PVA

Synthesis of AQ-PVA was prepared, as shown in the following reactionscheme:

This example and the following example illustrate methods and reagentsof the invention for making a non-sol-gel polymer-based matrix andcorresponding WE of the invention. Poly(vinyl alcohol) [Alfa Aesar,#41238, CAS 9002-89-5] ((0.29 g, 1 mole) was dissolved in a 1:1 mixtureof methanol and ethanol. 2-Bromomethyl anthraquinone (2 g, 6.6 mmole)was added with stirring until completely dissolved.Diethylaminomethylpolystyrene (1 eq.) was added as acid scavenger. Thereaction mixture was refluxed for 48 h. The solid formed was removed byfiltration, and the liquid fraction was evaporated to dryness.

Example 11 Preparation of AQ-PVA Working Electrode

0.1 g of the PVA-AQ solid product was dissolved in a mixture of 1.5 mLMethanol, 1.5 mL ethanol, and 1.0 mL DMF. The resultant solution wasapplied as a coating to prepared blank carbon substrate tips, air driedat room temperature, and heat treated at 150° C. for 1 h.

Samples of these working electrodes were tested on an Autolabpotentiostat with a pH 7 buffer standard at room temperature, with theresults shown in Table 3.

TABLE 3 Sample Code Peak position (V) Peak current (μA) X070 −0.486 39.4Y060 −0.485 36.3 X083 −0.483 26.2 Y067 −0.484 32.7 X082 −0.485 29.5 Y077−0.485 25.2

Many of the above Examples illustrate that strong, repeatable signalsare obtainable with sensors of the current invention. Their virtuallyconstant peak positions provide the basis for a reliable correlationbetween potential peak position and the pH value of the standard buffer.It was also seen that different ASM sensing surfaces differ in theirelectrochemical response, as shown in their peak positions.

What is claimed is:
 1. A matrix material comprising at least one of a sol-gel or other crosslinked polymer covalently attached to at least one of an analyte-sensitive material (ASM) and an analyte-insensitive material (AIM), wherein said sol-gel or other crosslinked polymer are formed by crosslinking an ASM silane precursor compound having the structure of Formula (IV):

wherein at least two of R₁, R₂ and R₃ are independently alkoxy, aryloxy, or methoxy, and the third is selected from the group consisting of alkyl, aryl, alkoxy, aryloxy, and methoxy; X₁ is —O— or a chemical bond; L is —(CH₂)_(n); Y₁′ is —OCONR₄—, —O—, —NR₄CO—, —COR₅—, —P(O)(OR₆)O—, —CO₂—, —O₂C—, —NO₂R₄, —CO₂NR₄—, —N═N—, —CONH—, —NH—, CO₂NH, NHR₄, or a chemical bond; ASM₁ is an ASM or an AIM; R₄, R₅ and R₆ are independently hydrogen, alkyl, or aryl; and n is 2-6.
 2. The matrix material of claim 1, wherein ASM₁ is selected from the group consisting of:


3. The matrix material of claim 1, wherein ASM₁ is selected from the group consisting of:

wherein R₂₀, R₂₁, R₂₂, R₂₃, R₂₄, R₂₅, R₂₆, R₂₇, R₂₈, R₂₉, R₃₀, R₃₁, R₃₂ and R₃₃ are independently hydrogen, —CO₂H, halogen,—OH, —NHR₄, —SO₂H, —R₅CO, —P(O)(OR₆)(OH), —N₃, —CN, alkyl, aryl or alkoxy with the proviso that least one substituent is —CO₂H, halogen,—OH, —NHR₄, —SO₂H, —R₅CO, —P(O)(OR₆)(OH), N₃ or —CN.
 4. The matrix material of claim 1, wherein said sol-gel or other crosslinked polymer formed by crosslinking the ASM silane precursor compound is selected from the group consisting of polyols, poly(vinyl alcohol), polysulfones, polyethersulfones, polyamides, polyimides, polyesters, vinyl polymers, polyphenylene sulfides, polysaccharides, cellulose, and derivatives, copolymers, blends, and composites of any of the foregoing or combinations of any of the foregoing.
 5. An electrode comprising or composed of a matrix material of claim
 1. 6. The electrode of claim 5 that is a working electrode.
 7. The electrode of claim 5 that is an analyte-insensitive electrode.
 8. The electrode of claim 5, wherein ASM₁ is selected from the group consisting of:


9. The electrode of claim 5, wherein said polymer is poly(vinyl alcohol).
 10. The electrode of claim 9, wherein said ASM or AIM is selected from the group consisting of:

wherein R₂₀, R₂₁, R₂₂, R₂₃, R₂₄, R₂₅, R₂₆, R₂₇, R₂₈, R₂₉, R₃₀, R₃₁, R₃₂ and R₃₃ are independently hydrogen, —CO₂H, halogen,—OH, —NHR₄, —SO₂H, —R₅CO, —P(O)(OR₆)(OH), —N₃, —CN, alkyl, aryl or alkoxy with the proviso that least one substituent is —CO₂H, halogen,—OH, —NHR₄, —SO₂H, —R₅CO, —P(O)(OR₆)(OH), N₃ or —CN.
 11. The electrode of claim 5, wherein said polymer is a cross-linked poly(vinyl alcohol) polymer, and said polymer is covalently attached to a redox active material comprising anthraquinone.
 12. The matrix material of claim 1, wherein said ASM silane precursor compound is selected from the group consisting of polyols, poly(vinyl alcohol), polysulfones, polyethersulfones, polyamides, polyimides, polyesters, vinyl polymers, polyphenylene sulfides, polysaccharides, cellulose, and derivatives, copolymers, blends, and composites of any of the foregoing or combinations of any of the foregoing.
 13. A matrix material comprising at least one of a sol-gel or other crosslinked polymer covalently attached to at least one of an analyte-sensitive material (ASM) and an analyte-insensitive material (AIM), wherein said sol-gel or other crosslinked polymer is formed by crosslinking an ASM silane precursor compound selected from the group consisting of:

and wherein at least one of said ASM silane precursor compounds is polymerized to form a sol-gel or other crosslinked polymer of Formula (XIX):

wherein n is one or more. 